Project Summary Lung cancer results in the largest number of cancer-related deaths in the United States. Non-small cell lung cancer (NSCLC) patient tumors overexpress NADPH oxidases that produce intracellular hydrogen peroxide (H2O2) and play an important role in lung cancer tumorigenicity. To date, our preliminary results demonstrate that NOX activity is functionally important for NCSLC transformation, escape from anoikis, phosphorylation signaling, and redox regulation of cysteines in several novel intracellular proteins. The fundamental goal of this proposal is to study the molecular details of NOX-dependent redox signaling in lung cancer biology. Aim 1 of the proposal will characterize the functional role of NOX isoforms overexpressed in lung patient tumors in lung cancer tumorigenicity. Aim 2 will examine the regulatory crosstalk between phosphorylation signaling and NOX activity by profiling NOX-dependent kinase activity. Aim 3 will quantitatively map the cysteine targets of NOX- derived H2O2 in NSCLC upon NOX inhibition. Investigating the role of NOX enzymes and redox signaling in lung cancer will shed light on orthogonal control mechanisms regulating cancer cell growth, transformation and evasion of anoikis. There is a strong link between RAS mutations, overexpression of NOX and increased ROS production. Therefore, investigating redox signaling in NSCLC may identify new regulators and therapeutic targets for intervention in lung cancer, especially RAS-driven tumors with no clear therapeutic option.